Abstract

The RecBCD enzyme of Escherichia coli participates in several aspects of DNA recombination and repair. It is essential to the main pathway of genetic homologous
recombination, where it contributes to the exchange of genetic material between homologous DNA molecules (i.e. conjugal recombination),
and to the recombinational repair of potentially lethal chromosomal double‐stranded breaks.

Keywords: RecBCD; recombination; hotspot; DNA repair; E. coli

Figure 1.

Initiation of homologous recombination by the coordinated activities of RecBCD enzyme and RecA protein. An in vitro model depicting recombination between a linear χ‐containing double‐stranded DNA (dsDNA) and a supercoiled plasmid is shown. (a–c) First, RecBCD enzyme enters at a dsDNA end and unwinds the duplex while preferentially degrading the strand corresponding to the 3′ terminus at the point of entry;
SSB protein binds the single‐stranded DNA (ssDNA) produced. (d,e) Upon recognition of χ, the 3′ to 5′ nuclease activity is attenuated and a weaker 5′ to 3′ nuclease activity
is activated on the opposite strand. Following the interaction with χ, RecBCD enzyme facilitates the loading of RecA protein
(to the exclusion of SSB protein) onto the ssDNA produced by continued translocation and unwinding of the DNA molecule. (e,f) This RecA protein–ssDNA filament then invades a homologous duplex DNA molecule, producing a recombination intermediate known as a joint molecule.

Figure 2.

Orientation dependence of χ recognition. For recognition to occur, the RecBCD enzyme must approach the χ site from the 3′
side, as depicted. Recognition results in attenuation of the 3′ to 5′ nuclease activity of the enzyme with the final cleavage
event occurring near the 3′ end of χ. Arrows denote the positions at which the final cut may occur, with corresponding thicknesses
indicating the relative frequencies at each position. Adapted from Kowalczykowski et al. and Taylor and Smith .

Figure 3.

Recombinational repair of a collapsed replication fork. (a) Unreplicated E. coli chromosome. (b) Replication is bidirectional with two replication forks initiating at oriC and progressing in opposite directions around the chromosome; a lesion in the DNA template blocks progression of one replication
fork. (c) The blockage causes detachment of one arm and collapse of the replication fork resulting in a dsDNA break. (d) RecBCD enzyme enters at the dsDNA end, degrading until reaching a χ site; enzymatic modification of RecBCD enzyme occurs and the facilitated loading of RecA
protein follows. (e) RecA protein promotes strand invasion of the ssDNA substrate into the homologous duplex, recreating a template for replication as shown in the shaded circle. (f) The replication
fork is reassembled and replication of the chromosome resumes. The Holliday junction formed by the strand exchange event is
resolved by the specific resolvases of E. coli.

References

Anderson DG and
Kowalczykowski SC
(1997a)
The recombination hot spot χ is a regulatory element that switches the polarity of DNA degradation by the RecBCD enzyme.
Genes and Development
11: 571–581.